Summary: Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays for their differentiation and the existence of atypical strains. The phylogeny of twelve Campylobacter species was studied based on partial (1020-bp) gyrB gene (DNA topoisomerase beta-subunit gene) sequences. The topology of the resulting phylogenic neighbor-joining tree based on the gyrB gene was similar to that of the topology of a previously reported phylogenic tree based on the 16S rDNA gene. However, gyrB provides a better resolution than the 16S rDNA gene for Campylobacter species with interspecies sequence similarities ranging from 58.3 to 89.2%. A universal primer set, designed to amplify a 960-bp fragment of the gyrB gene in Campylobacter spp., was developed and used for PCR restriction fragment length polymorphism (PCR-RFLP) of 19 strains representing twelve Campylobacter species, including C. jejuni subsp. jejuni, C. coli, C. concisus, C. curvus, C. showae, C. mucosalis, C. fetus, C. hyointestinalis, C. sputorum biovar sputorum, C. helveticus, C. upsaliensis, and C. lari. Digestion of the 960-bp fragment with the restriction enzymes DdeI, XspI, or the combination of MboI and HindIII, as a double digestion, resulted in unique digest patterns for all twelve Campylobacter species. In addition, PCR assays were developed using species-specific primer sets for amplification of regions of the gyrB gene specific for each Campylobacter species, yielding products ranging in size from 86 to 493 bp. Specificity testing using DNA from Campylobacter spp., Arcobacter spp., Helicobacter spp., and other bacterial genera showed that the Campylobacter species-specific primer sets were highly specific for the respective target species. In conclusion, PCR-RFLP analysis, and PCR using the species-specific primer sets based on the gyrB gene provide valuable tools for rapid detection and unambiguous identification of the majority of Campylobacter species.